Abstract
Background: Numerous studies have proven that protein tyrosine phosphatase type IV A member 3 (PTP4A3) plays a significant role in local lymph node metastasis from colorectal carcinoma. The aim of the current study was to assess PTP4A3 expression in colorectal carcinomas and lymph nodes in correlation with chosen anatomoclinical parameters. Patients and Methods: Forty-nine patients were analyzed with respect to age, tumor location, pTN stage and local lymph node involvement. The level of PTP4A3 expression was determined immunohistochemically using mouse monoclonal anti- PTP4A3 antibody (monoclonal antibody 3B6, anti-human PTP4A3 from Attogen Biomedical Research, USA). Results: Positive reaction for PTP4A3 in the main tumor mass was observed in 9/49 (18.4%) cases, whereas in metastatic lymph nodes in 22/24 (91.6%) cases. Statistical analysis showed no correlation of PTP4A3 expression in the main tumor mass with such clinical parameters as age, location, histological type or lymph node involvement. Moreover the expression of PTP4A3 in the main tumor mass was not associated with the expression of this protein in lymph node metastases. However, there was a statistically significant correlation between lymph node involvement and PTP4A3 expression in lymph node metastases. Conclusion: The role of the PTP4A3 has not yet been fully elucidated. Our finding indicate PTP4A3 is strongly expressed in colorectal carcinoma metastases of the lymph nodes although the significance of this is unclear.
Colorectal carcinoma is one of the most common causes of death due to malignancy, its incidence showing a growing tendency. Tumor stage is the major prognostic factor and colorectal carcinoma frequently metastasizes to local lymph nodes, liver and lungs. The ability of cancer cells to disseminate from primary tumors to lymph nodes as well as to the nearest and distant tissues and organs is an essential feature of malignant neoplasms and the main cause of therapeutic failures. Formation of metastases is a complex and multistage process involving proteolysis, mobility and migration of cells, proliferation and angiogenesis. Cancer cells released from the primary tumor have to invade the adjacent tissues, permeate lymphatic or blood vessels, migrate through the vascular walls to the adjacent tissues, where they eventually settle, proliferate and induce angiogenesis, forming metastatic foci. Early detection of metastases offers a good chance of a cure. A recent study indicated that the protein formerly known as phosphatase of regenerating liver-3 (PRL-3) newly named protein tyrosine phosphatase type IV A member 3 (PTP4A3) could be a marker for metastasis (1).
The PTP family comprises a large group of enzymes taking part in one of the most important reactions in the cell, i.e. dephosphorylation of tyrosine residues that are involved in activation or inactivation of enzymes. In this way, they regulate a number of cellular processes, both physiological and pathological, including: cell growth, differentiation, cycle or neoplastic transformation. Non-classic representatives of this family include three closely related phosphatases (PTP4A1-PTP4A3) with a unique COOH terminal group. All these proteins have at least 75% of their amino acid sequences in common. PTP4A1 is localized primarily in the brain and muscles, PTP4A2 in skeletal muscles and PTP4A3 in cardiac muscle and skeletal muscles (2). PTP4A3 protein has an active site with the signature motif CX5R, where Cys104 is an enzymatic nucleophile and Arg110 binds to phosphate groups on phosphotyrosine (3). The PTP4A3 molecule contains C-terminal consensus sequence for prenylation, and it is localized in membranes and intracellular structures when prenylated and in the nucleus when it is not. The role of PTP4A3 is not well known. Matter et al. (4) have suggested that PTP4A3 may play a role in the regulation of intracellular calcium transmitters induced by angiotensin II. Wu et al. (5) discovered the presence of PTP4A3 in cellular membrane components in the mitotic phase specifically in metaphase. This location may suggest that PTP4A3 is engaged in cell cycle regulation. The protein also plays a significant role in the induction of angiogenesis by recruiting endothelial cells from the circulating blood and in the formation of microcirculation (6). However, abnormal regulation of tyrosine phosphorylation and dephosphorylation may also lead to neoplastic formation. PTP4A3 protein expression has been detected in cancer of the ovaries (7), breast (8), stomach (9) and large intestine (1).
The aim of the present study was to assess the expression of PTP4A3 in colorectal carcinomas and in lymph nodes in correlation with chosen anatomoclinical parameters.
Patients and Methods
The study was conducted in a group of 49 patients treated surgically due to colorectal carcinoma in the Department of Surgery, the J. Śniadecki Hospital in Bialystok. Sections, 4 μm thick, were cut from paraffin blocks and stained with hematoxylin-eosin (H+E). The routine histopathological assessment was performed for histological type, malignancy grade (G), infiltration depth and the presence of lymph node metastases.
Immunohistochemical analysis. Formalin-fixed and paraffin-embedded tissue specimens were cut on a microtome into 4 μm sections. The sections were deparaffinized in xylene and hydrated in alcohol. To visualize the antigen, the sections were heated in a microwave oven for 15 min in a citrate buffer (pH 6.0). They were incubated with 0.5% hydrogen peroxide solution in methanol in order to block endogenous peroxidase. Incubation was then performed with mouse monoclonal antibody against human PTP4A3 (monoclonal antibody 3B6; Attogen Biomedical Research, USA) over night at 4°C. The secondary reaction was carried out using biotinylated anti-mouse antibody conjugated with horseradish peroxidase (LSAB2, DAKO, Poland). A colour reaction for peroxidase was developed with the chromogen diaminobenzidine (DAKO). Protein expression was determined using a semiquantitative method and assessed as positive (reaction visible in >5% of tumour cells) or negative (lack of reaction, or reaction present in <5% of cells). Reactions were assessed in at least 500 cancer cells in each tissue specimen under a light microscope (×400).
Statistical analysis was conducted using Fisher's test and χ2 analysis. A p-value <0.05 was considered statistically significant.
Results
The study group consisted of 49 patients with colorectal carcinoma pT3G2. Twenty-four patients (49%) had lymph node metastases and four (8%) distant metastases. Cytoplasmic PTP4A3 expression in the main mass of a tumor and no reaction in normal mucosa has been presented in Figure 1. Positive reaction for PTP4A3 protein in the main tumor mass was observed in 9/49 (18.4%) cases, whereas in metastatic lymph nodes, in 22/24 (91.6%) cases. Statistical analysis did not show any correlation between PTP4A3 expression in the main mass of tumor and such clinical parameters as age, location, histological type, the presence of metastases in lymph nodes and distant metastases. The relationship between PTP4A3 expression in the main tumor mass was not correlated with PTP4A3 expression in lymph nodes. Nor was PTP4A3 positivity in lymph nodes correlated with other factors. The relationship between the presence of lymph node involvement and PTP4A3 expression in the lymph nodes was, however, statistically significant (p<0.0001) (Table I).
PTP4A3 expression in the main tumor mass and in metastatic lymph nodes in association with anatomoclinical parameters.
Discussion
The current study objective was to analyze the role of PTP4A3 expression in colorectal cancer in correlation with the presence of lymph node involvement. The expression of PTP4A3 in the main tumor mass was significantly lower than that in the lymph node metastases. Saha et al. (1), who conducted a molecular study on PTP4A3 gene expression, noticed that it was high in 18/18 metastases, whereas low in non-metastasizing carcinomas and in normal epithelial tissue. Interestingly, in 3/12 metastases they found duplicate copies of the PTP4A3 gene localized in a small amplicon at chromosome 8q24.3, which suggests the significance of the PTP4A3 gene in colorectal carcinoma. In addition, Buffart et al. (10) compared the number of PTP4A3 gene copies in the primary tumor of colorectal carcinoma with and without metastases to the liver. These observations seem to suggest that metastasis may be associated with the genetic profile of the primary tumor and that amplification of the PTP4A3 gene is a mechanism leading to protein over expression. They also found high expression of PTP4A3 in the vascular system of the tumor irrespective of the source of the neoplasm. Bardelli et al. (11) observed high PTP4A3 mRNA expression in colorectal cancer metastases to other organs (in 10/11 metastases to the liver, 6/7 to the lungs, 4/4 to the brain and in 3/3 to the anus), whereas there was no or only low expression of the protein in metastases to these organs from other types of carcinoma (pancreatic, gastric, esophageal) and in non-metastasizing colorectal carcinoma. Moreover, they discovered that the expression of PTP4A3 was higher in over 95% of metastases as compared to the increased number of PTP4A3 gene copies in colorectal cancer (1), thus showing that PTP4A3 gene amplification is not the major cause of the protein overexpression. It was also demonstrated that PTP4A3 overexpression was not due to the presence of neoplastic metastatic cells in the liver. Kato et al. (12) observed similar relations in PTP4A3 protein expression in colorectal carcinomas and their metastases. They also showed that PTP4A3 overexpression is associated with the mobility of neoplastic cells but had no effect on their proliferation.
Immunohistochemical staining. A, Lack of staining for PTP4A3 in normal mucosa. B, Colorectal cancer tissue showing cytoplasmic staining for PTP4A3. Original magnification, ×400.
In an immunohistochemical study concerning PTP4A3 expression, Peng et al. (13) observed high overexpression of this protein in colorectal cancer metastasis to local lymph nodes (53.7% cases) and also in liver metastases (66.7% of cases), whereas there was low protein expression in non-metastasizing tumor (23.9% of cases) as well as in normal colorectal epithelium (7.1% of cases). The expression of PTP4A3 protein in the main tumor was found to correlate with lymph node involvement, but not with the presence of distant metastases. In our study, the expression of PTP4A3 in lymph node metastases was high in 22/24 cases, but the protein expression in the main tumor mass did not correlate with the presence of metastases, in contrast to the finding of Wang et al. (14).
Although our findings show no correlation between PTP4A3 expression and colorectal cancer metastasis per se, its high expression in lymph node metastases is of significance. PTP4A3 involvement in colorectal cancer metastasis needs to be further investigated.
Acknowledgements
We thank Dr. Jerzy Hapanowicz, Department of Surgery Hospital in Bialystok for cooperation.
- Received May 11, 2009.
- Revision received August 30, 2009.
- Accepted September 1, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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